The current understanding of the regulation of iron metabolism is based on the biology of a number of critical proteins, which include transferrin, transferrin receptors, ferritin, iron regulatory proteins, divalent metal transporter 1, ferroportin, and hepcidin (Non-patent Documents 1 to 4). Among these factors, plasma transferrin and ferritin are generally measured in the laboratory as an indicator of the total iron binding capacity and overall iron storage, respectively. The peptide hepcidin, which is produced by the liver, controls plasma iron levels by regulating the absorption of food iron from the intestine as well as the release of iron from macrophages. Furthermore, hepcidin is also an acute-phase reactant with antimicrobial activity induced by inflammation (Non-patent Documents 5 to 7). Most studies that confirm the role of hepcidin in iron metabolism, inflammation, anemia, and hypoxia were performed in vitro or using experimental mice (Non-patent Documents 5 and 8); therefore, its role in human diseases is unclear.
Renal anemia in hemodialysis patients can be clinically alleviated by the administration of human recombinant erythropoietin (Non-patent Document 9); however, the exact mechanism of iron metabolism in these patients is largely unknown. In several clinical studies, the amount of hepcidin was estimated based on the levels of prohepcidin in urine (Non-patent Documents 6 to 10 and 13) or serum (Non-patent Documents 14 to 16) or based on the levels of mRNA expression in the liver (Non-patent Document 10); however, it has been reported that serum prohepcidin concentration does not clearly correlate with any red cell indices or with iron status (Non-patent Document 14) and that the concentration of prohepcidin may increase because of its accumulation. In addition, prohepcidin, which is measured by enzyme-linked immunosorbent assay (ELISA), is not bioactive and no specific function has been identified. Hepcidin-20, -22, and -25, which are cleaved from prohepcidin by convertases, are the active forms of prohepcidin (Non-patent Document 6); however, there are few reports on the quantitative evaluation of the bioactive forms of hepcidin, primarily because of difficulties involved in the development of specific antibodies against these bioactive forms of hepcidin, which have compact folded structures (Non-patent Document 17).
Hepcidin, which is a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. To date, some diagnostic methods using hepcidin as an indicator have been reported (Patent Documents 1 to 4); however, no reliable and practical method for the measurement of the bioactive forms of hepcidin in serum has been developed.
The SELDI-based ProteinChip System® (Ciphergen Biosystems, Inc., Fremont, Calif., USA) array technology has been successfully used to detect relevant biomarkers in a wide variety of diseases, which include immunological conditions, cancer, neurological conditions, cardiovascular conditions, and diseases of the lacrimal gland (Non-patent Documents 18 to 21). SELDI technology is based on a classical solid-phase extraction chromatography method combined with direct laser desorption/ionization mass spectrometric detection and requires minimal amounts of biological fluid, without pretreatment. This technology enables the evaluation of the subtle differences between disease and control states in the expression of individual proteins or groups of proteins in various fluids, which include serum, urine, tears, and cerebrospinal fluid. Furthermore, it has a number of advantages, which include high-throughput capability, very high sensitivity for the detection of proteins in the picomole to femtomole ranges, high resolution for low-molecular-weight proteins (i.e., below 20 kDa), and facility of operation.
Generally, hepcidin-20 is known as an antimicrobial peptide (Non-patent Document 22), and hepcidin-25 is known as an iron-regulatory peptide (Non-patent Document 23).
In recent years, the use of the above-mentioned SELDI-TOF MS has enabled semi-quantitative measurement of hepcidin-20 and -25 in the serum, and it has been reported that hepcidin-20 and -25 of hemodialysis patients show significant correlation with the level of serum ferritin, and that hepcidin-25 accumulates in the serum of hemodialysis patients. Furthermore, it has been reported that active hepcidin, particularly hepcidin-25, may be contributing to the etiology of renal anemia (Non-patent Document 24).
However, there are no reports on the association of hepcidin, or particularly the association of hepcidin-20, with acute ischemic diseases.
Documents of related prior arts for the present invention are described below.    Non-patent Document 1: Andrews N C. Best Pract Res Clin Haematol. 2005; 18:159-169.    Non-patent Document 2: Philpott C C. Hepatology. 2002; 35:993-1001.    Non-patent Document 3: Fleming R E. Curr Opin Gastroenterol. 2005; 21:201-206.    Non-patent Document 4: Ganz T. Blood. 2003; 102:783-788.    Non-patent Document 5: Pigeon C, Ilyin G, Courselaud B, et al. J Biol Chem. 2001 16; 276:7811-7819.    Non-patent Document 6: Park C H, Valore E V, Waring A J, Ganz T. J Biol Chem. 2001; 276:7806-7810.    Non-patent Document 7: Krause A, Neitz S, Magert H J, et al. FEBS Lett. 2000; 480:147-150.    Non-patent Document 8: Nicolas G, Chauvet C, Viatte L, et al. J Clin Invest. 2002; 110:1037-1044.    Non-patent Document 9: National Kidney Foundation. Am J Kidney Dis. 1997; 30(Suppl 3):S192-240.    Non-patent Document 10: Detivaud L, Nemeth E, Boudjema K, et al. Blood. 2005; 106:746-748.    Non-patent Document 11: Kemna E, Tjalsma H, Laarakkers C, Nemeth E, Willems H, Swinkels    D. Blood. 2005 Jul. 19; [Epub ahead of print]    Non-patent Document 12: Nemeth E, Rivera S, Gabayan V, et al. J Clin Invest. 2004; 113:1271-1276.    Non-patent Document 13: Nemeth E, Valore E V, Territo M, Schiller G, Lichtenstein A, Ganz T. Blood. 2003; 101:2461-2463.    Non-patent Document 14: Taes Y E, Wuyts B, Boelaert J R, De Vriese A S, Delanghe JR. Clin Chem Lab Med. 2004; 42:387-389.    Non-patent Document 15: Kulaksiz H, Gehrke S G, Janetzko A, et al. Gut. 2004 May; 53(5):735-743.    Non-patent Document 16: Dallalio G, Fleury T, Means R T. Br J. Haematol. 2003; 122:996-1000.    Non-patent Document 17: Hunter H N, Fulton D B, Ganz T, Vogel H J. J Biol Chem. 2002; 277:37597-37603.    Non-patent Document 18: Petricoin E F, Ardekani A M, Hitt B A, et al. Lancet. 2002; 359:572-577.    Non-patent Document 19: Sanchez J C, Guillaume E, Lescuyer P, et al. Proteomics. 2004; 4:2229-2233.    Non-patent Document 20: Stanley B A, Gundry R L, Cotter R J, Van Eyk J E. Dis Markers. 2004; 20:167-178.    Non-patent Document 21: Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I. J Proteome Res. 2005; 4:820-825.    Non-patent Document 22: Krause A, FEBS LETT. 2000 Sep. 1; 480(2-3):147-50.    Non-patent Document 23: Park C H, J Biol Chem. 2001 Mar. 16; 276(11):7806-10. Epub 2000 Dec. 11    Non-patent Document 24: Tomosugi N, Blood. 2006 Aug. 15; 108(4):1381-7. Epub 2006 Apr. 18.    Patent Document 1: Japanese Patent Application Kokai Publication No. (JP-A) 2005-134387 (unexamined, published Japanese patent application)    Patent Document 2: US 2004/0096987 A1    Patent Document 3: US 2004/0096990 A1